Chondrocytes are acutely sensitive to physical cues across multiple length scales ranging from compression to extracellular matrix stiffness. However, in osteoarthritis, the physical and biological properties of articular cartilage are disrupted through mechanisms that are coupled but unclear. Although changes in extracellular matrix stiffness are among the earliest detectable signs of osteoarthritis, the extent to which these physical changes in the chondrocyte microenvironment contribute to the loss of chondrocyte homeostasis is not fully understood. Cells sense and respond to physical cues in their microenvironment through integrin-rich focal adhesions and actomyosin-generated cytoskeletal tension. Changes in cytoskeletal tension affect cell signaling and gene expression, which in turn, regulate basic cellular processes such as proliferation and differentiation. For example, changes in cytoskeletal tension drive TGF-induced Smad3 phosphorylation and translocation to control chondrogenic gene expression. Although cytoskeletal tension modifies the cellular response to signaling by several growth factor signaling pathways, the molecular mechanisms responsible for this sensitivity remain unclear. Therefore, the goal of this project is to identify novel molecular mechanisms by which cytoskeletal tension regulates TGF signaling and the role of these mechanisms in the well-documented response of cartilage to multi-scale physical cues. To achieve this goal, the proposed research will test the hypothesis that physical cues regulate chondrocyte behavior by inducing changes in cytoskeletal tension, which, in turn, influences growth factor receptor localization and function. Aim 1 will identify mechanisms by which cytoskeletal tension alters the cellular response to growth factors. Preliminary data suggest that cytoskeletal tension regulates TGF signaling at the level of the cell membrane, regulating physical and functional interactions among TGF receptors, integrins, and their effectors. New molecular tools, super-resolution quantitative imaging, and biochemical approaches will be employed to pursue this possibility. Aim 2 will determine the extent to which cytoskeletal tension is a common mechanism by which chondrocytes respond to diverse physical cues. Using cell-seeded 3D constructs, Aim 2 extends mechanisms identified in Aim 1 to understand their role in the maintenance or loss of chondrocyte homeostasis by TGF signaling. The completion of this research will yield new molecular mechanisms by which cells integrate physical and biochemical cues. This contribution is significant because it will advance the identification of molecular targets that uncouple the physical degeneration of cartilage, due to injury or disease, from the loss of chondrocyte homeostasis, to prevent or block osteoarthritis.